Synthesis of a ligand-quencher conjugate for the ligand binding study of the aryl hydrocarbon receptor using a FRET assay

Yu Wang, Dazhou Yang, Abraham Chang, William K. Chan, Bin Zhao, Michael S. Denison, Liang Xue

Research output: Contribution to journalArticle

2 Scopus citations

Abstract

The aryl hydrocarbon receptor (AhR) is a transcription factor that induces the adaptive responses upon binding to a wide range of exogenous chemicals in cells. The traditional method for studying AhR ligands is to determine their effect on transcriptional activities (e.g., luciferase expression) in cultured cells. In this paper, we sought to investigate the feasibility of studying the ligand binding with the AhR using a Förster resonance energy transfer (FRET) assay-a novel approach. Two conjugates (βNFQ and 8) containing β-naphthoflavone and DABCYL (a quencher) with different linkers were therefore synthesized for evaluation. The luciferase expression and green fluorescent protein (GFP) expression assays showed that βNFQ is a partial AhR agonist. Simultaneous incubation of cultured cells containing expressed GFP-AhR fusion protein with βNFQ and a known AhR ligand 3-methylchloranthrene reduced the fluorescence of GFP-AhR to a lesser extent, suggesting that the reduction in fluorescence resulted from the competitive binding of ligands to GFP-AhR. Although the fluorescent quenching by βNFQ is modest, our results suggest that FRET could be a valuable tool for identifying AhR ligands with the use of a more efficient ligand-quencher.

Original languageEnglish (US)
Pages (from-to)711-721
Number of pages11
JournalMedicinal Chemistry Research
Volume21
Issue number6
DOIs
StatePublished - Jun 2012

Keywords

  • β-Naphthoflavone
  • Aryl hydrocarbon receptor ligands
  • Förster resonance energy transfer
  • Green fluorescence protein

ASJC Scopus subject areas

  • Pharmacology, Toxicology and Pharmaceutics(all)
  • Organic Chemistry

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